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In this study, nano drug delivery system based on graphene oxide and reduced graphene oxide- polyglycerol hybrids were constructed. Functionalization of nano graphene oxide and reduced graphene oxide was accomplished through noncovalent interaction between the π conjugated system of graphene materials and the aromatic segment in the focal point of polyglycerol polymer. Polyglycerol is a hydrophilic and biocompatible polymer that its conjugation with graphene materials was increased the colloidal stability and decreased the nonspecific interaction of graphene materials. Curcumin as an anticancer hydrophobic natural drug with low systemic bioavailability was simply loaded on these nanohybrids via π-π stacking force between the π conjugated systems of graphene materials and curcumin. Result showed that loading capacity of curcumin for reduced graphene oxide hybrid (49%) is higher than graphene oxide hybrid due to restored of π conjugated system in reduced graphene oxide. Anticancer efficiency of these drug hybrids was investigated by MTT assay. Results showed that these drug carriers have sufficient biocompatibility. Also these nano drug delivery systems showed a cytotoxic effect that was comparable to that of free curcumin. The reduced graphene oxide hybrid is preferred for delivery of curcumin due to its higher loading capacity that can provide efficient dose of drug in low level of carrier

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Background: Rheumatoid Arthritis (RA) is the most common chronic inflammatory erosive joint disease with the worldwide distribution of approximately 0.5-1%. Molecular methods can be very sensitive in the early stage diagnosis of the disease and therefore prevention the late complications and disabilities. Etiology of RA, an autoimmune disease, is not exactly known but immunologic and genetic factors seem to play important roles in the pathogenesis of the disease. Genetic factors such as Human Leukocyte Antigens (HLA) are responsible for many autoimmune diseases; therefore we decided to look for a correlation between RA and the presence of specific HLA-DQB1 alleles as a possible genetic markers. Methods: The genomic DNAs from the whole blood samples of 25 patients with RA and 86 normal individuals (Control group) were extracted by salting out method. The genomic DNA was amplified by PCR-SSP technique. HLA-Typing was done by this method after optimizing the PCR reaction for each allele. In this procedure seven serological subclasses of HLA-DQB1 can be detected. Results: Comparing the results between the patients and the controls suggests a significant increase in the frequency of HLA-DQ5 (*0501-*0504) and HLA-DQ8 (*0302, *0305) alleles in the patient group. The P-values were respectively 0.033 and 0.007 (P<0.05)(DQ5 and DQ8 positive individuals in patients were respectively 56% and 24% compared with 31.4% and 5.8% in healthy people). The relative risk for these alleles was evaluated higher than 1. Conclusions: The results suggest that DQ5 and DQ8 are the dominant HLA-DQB1 alleles that are associated with the susceptibility to RA. These alleles are probably important in increasing the risk of developing RA disease in Northeastern Iran and we can use them as molecular markers for diagnosis of RA.

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Colorectal cancer (CRC) is one of the most common cancers worldwide. Understanding of the tumor behavior, in a much closer look, at the molecular level, results in a more effective treatment and accurate prognosis of the disease. From among various genes altered in colorectal cancer K-ras is assumed to be of diagnostic and prognostic significance. K-ras mutations are believed to be a critical event in colorectal oncogenesis. Previous studies have demonstrated that 40% (20-50%) of CRCs harbor a mutant allele of K-ras oncogene. The mutations are limited to codons 12, 13, and 61 of the gene, with a great incidence at codon 12. The localization of mutations has given mutated K-ras an advantage of sensitive and simple detection over APC or p53 in which mutations are spread in their whole DNA sequence. To determine the incidence of K-ras mutations in CRC in Iran, compared with other countries, DNA was isolated from a random collection of 55 colorectal carcinoma samples, and codon 12 K-ras mutations were detected by RFLP. K-ras mutations in sporadic colorectal cancer in Iran are relatively frequent, with an incidence of 65%. This may be attributed to variation in methodology and to characteristics of the population studied such as differences in genetic background and variability in environmental factors and epidemiologic parameters such as diet, social lifestyle status, and other parameters that could be specific to the Iranian population. Correlation between the presence of codon 12 mutation and various clincopathological parameters was also investigated. A significant correlation was found with poor tumor differentiation of tumor samples. This places much emphasis on the role of promotion of differentiation as the most prominent effect of Ras.

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Current investigations indicate different in vitro infection patterns for Oka and Dumas strains. Infection of cells in culture with Dumas strain produces lower number of infectious particles while the Oka strain is highly infectious in vitro. It is postulated that weak expression of the replication genes in Dumas strain may be the reason for the attenuated phenotype. The objective of this study was to analyze the sequences of the promoter region in two of the VZV replication genes, 16 and 52 by studying the level of expression of a reporter gene. For this purpose, primers were designed from VZV published sequences to amplify the promoter regions of both genes using Polymerase Chain Reaction (PCR) technique. The amplicons were cloned in a lacZ reporter vector. Cotransfaction of 52-Oka reporter plasmid with virus major trans-activator (IE62) in Huh7 cells showed that the presence of 52-Oka promoter was up regulated by the IE 62 trans-activator in a dose dependent manner resulting in ß-gal levels approximately 4-fold higher than those observed with 52-Dumas promoter and 10-fold higher than basal levels. In addition cotransfection of 16-Oka reporter plasmid did not show any significant change in activity in comparison with 16-Domus-reporter plasmid. Sequence determination of the promoter region in gene 52 indicated differences in 3 nucleotides in Dumas strain compared to Oka strain while no change was observed in the promoter sequences of gene 16 of the two strains. It is hence postulated a relationship between mutations in the Dumas promoter of replication genes, and the lower infectivity of the Dumas strain.

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Aims: The induction of artificial over-expression of miRNAs is an appropriate approach to more effective cell differentiation. The significant role of microRNA-1(miR-1) has been reported in the development and differentiation of cardiac cells. Lentivirus is an effective vector for stable cell line production. The aim of this study was the production of recombinant HEK293T with miR-1 overexpression as a biological model for cardiac studies.
Materials & Methods: In this experimental study, HEK 293T cells were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and L-glutamine 2mM and Penicillin-Streptomycin 1X in incubator medium. After cloning of miR-1 gene, recombinant clones were selected and the recombination was confirmed by sequencing. The miR-1 carrying vector and auxiliary vectors were packaged in the HEK293T to produce the recombinant virus. The infection of HEK293T by recombinant virus was performed in order to achieve stable cell line. Then, GFP fluorescent marker evaluated the efficiency of transfection and effective virus dilution. Finally, the alteration in expression level of miR-1 was assessed by qPCR. Data analysis was performed by comparing the threshold cycle and Pfaffl method.
Findings: The most GFP expression was detected in transfected cells by 150 micromole dilution. GFP fluorescent marker facilitated optimization and purification of recombinant cells. qPCR investigation demonstrated the significant increase in expression of miR-1 in transfected cells in comparison to controls.
Conclusion: The stable recombinant HEK293T miR-1 over-expressing cell line in lentivirus can be utilized as a suitable biological model for investigation of cardiac evolution and development processes.

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Objective: DNA markers are one of the most important indicators for estimating Molecular weight of DNA samples, although it used in widespread medical and research laboratories. These markers are very divers and have been prepared in different manners and from different sources of DNA. But unfortunately, DNA markers haven't been made in our country and all of the markers that we use are made in a foreign country. The aim of this research is settings a suitable technology to produce this product in the lab. Material and Methods: With this aim, we used two different strains of lambda: c1857sam7 and EMBL3A both of which are lytic phages as a DNA source. These were grown in the suitable host, after plaque appearance on the bacterial lawn, suitable titer for phage collecting was determined. We also optimized plasmid purification method for extraction of pBR322, pUC18 and recombinant VZV plasmid DNA and designed fragments in the markers have been constructed by digesting these DNAs with variant enzymes. Results: In this study, we made seven DNA markers out of which four of them were made for the first time in the world (/Hind III/BamH1, /Hind III/EcoR1, Sam2, Sam1) and although foreign models of three of them exist but they were made in our country for the first time (/Pst I, /Hind III, pBR332/MspI). Conclusion: The other goal of this study was to determining the best conditions for maintaining and preserving these markers in the lab which was successfully performed.

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Objective: The ITPA gene is responsible to remove free deaminated purine nucleotides of ITP, dITP and XTP from nucleotide pool of the cells. It seems that dysfunction in its activity, not only can increas the base substitution mutations frequency but also can works as a contrived factor to creating instability in genetic materials of the cells. There are several reports about the existence of structural and numerical genetic instability in the K562 cell genome. In this research, we examined the expression of ITPA gene as a possible contrived factor in observed genetic instability of this cell line. Materials and Methods: To evaluate the expression of target gene semi-quantitative RT-PCR technique was used. Then to examine the functionality of gene products, its cDNAs were cloned and their sequences were determined. Their proteins products were predicted using available bioinformatics soft wares and the results were compared. Results: The result of structural prediction of second mRNA showed that it has ability to encode a protein which has inability in substrate binding and also in its normal enzymatic activity. With regard to the fact that enzymatic activity of protein is dependent on the dimer formation, the function of hetero-dimer enzyme is changed. Therefore the catalytic activity of ITPase is predicted to be abnormal and it can be considered as a contrived factor for creating genetic instability in K562 cell line. Conclusion: The study of gene expression showed that ITPA gene is expressed in moderate level compared to GAPDH expression as an internal control in K562 cell. Two types of transcripts were detected in this line. One of them was the normal product of splicing process of primary hnRNA, but the second one contained a 51 nucleotides deletion in the mRNA coding region. It seems, this transcript is the product of a rare splicing process in this line.

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Objective: E1A oncoprotein of adenovirus type 5 is a regulatory factor which controls transcription of other adenovirus genes. This protein promotes both viral genom replication and host cell transformation by altering the function of certain important cellular proteins such as p21 and Rb. The aim of the present study was to constantly reduce the expression of E1A gene in HEK 293 cell line by RNAi technique in order to analyse the effects of this suppression on these cells. Materials and Methods: The U6 promoter and shRNA regions from the control and E1A specific siRNA coding plasmid as pSP-81 and pSP81-E1A were subcloned into pcDNA3.1. Then these constructs were transfered into the HEK 293 cancerous cells using lipofection method and successfully transfected cell colonies were selected based on neomycin antibiotic resistance. Changes in E1A gene expression were analysed by RT-PCR technique after selection process. Results: Final analysis showed no obvious difference in E1A gene expression level in the suppresed and control groups, upon transfection with the constructed plasmids. In order to examine the possible influence of cloning procedure on the function of U6 promoter, cells were transfected with Dr. Hacker’s original plasmids, but no inhibition of E1A gene expression was observed again. The results of sequencing revealed existence of a mutation in the siRNA target region for the E1A gene sequence. Conclusion: These results illustrated that no considerable suppression has been occurred by repeating Dr. Hacker’s expriment, even with application of very effective lipofection method. To examine the sequence of the E1A gene, the PCR product of the 13s region of the gene was sequenced. Sequencing revealed existence of a point mutaion in the siRNA target region. It seems that observed impaired interference could be attributed to this mutation in the E1A gene of the studied cells.

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Objective: β-thalassemia is caused by absence or reduction of β-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and β-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and β-globin gene for transfer to the target cells for gene therapy of β-thalassemia. Materials and Methods: HS2, HS3, HS4 segments (mini LCR) and β-globin gene with 5΄ and 3΄ UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids (Plp1, Plp2, Plp/VSVG) were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. Results: The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random integration of construct into the genome was evaluated. Conclusion: The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene.

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Objective: Chronic Myeloid Leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells which results in increase of myeloid cells, erythroid cells and platelets in the peripheral blood and hyperplasia in bone marrow. Investigations have shown different types of BCR- ABL variants in these patients. In Iran only 3 types of these variants have been identified because most of the clinical laboratories usually use only few sets of primers which can not detect all types of the variants simultaneously. In this study we developed a method with which all types of variants in chronic Myeloid leukemia patients can be recognized. Materials and Methods: blood samples from 100 persons who were under treatment or diagnosis for CML were received from Clinical laboratory. RNA was extracted from 600 µl of each sample using Roche Commercial kit and converted to cDNA by reverse transcriptase enzyme the cDNAs were analyzed for BCR- ABL variants using two sets of primers. All samples were also studied by RT- Multiplex Nested PCR method. Results: RT-Multiplex PCR could detect BCR-ABL in all samples which were positive for these Fusion Gene mRNA. From 100 collected samples 46 percent were positive and 54 percent were negative by RT-Multiplex PCR method and 44 percent were positive and 56 percent were negative by RT-Multiplex Nested PCR method. Conclusion: By using one step PCR we detected more variants of BCR- ABL in one tube at shorter time and lower cost. This method showed 100 percent specificity and can further be improved by taking more samples and also real time PCR for quantitative analysis.

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Aims: The accumulation of free radicals in the body leads to damages to cellular biopolymers through oxidative stress. Due to the increasing proliferation of heavy metals in soil and water environments, finding efficient methods for diagnostic detection and measurement of heavy metals in contaminated environments is very important. Cell-based biosensors can produce a measurable signal in response to specific chemical or physical agent in their environment. In this study, stable hepatoma Huh7 ARE-reporter cell line was developed containing luciferase gene with the aim of monitoring lead toxicity. This biosensor is reported to be able to detect lead by expressed signal which is measurable. The luciferase assay and Real time-PCR were performed.
Materials and Methods: In this experimental research, the Huh7-1x-ARE-luc was stably transferred in to the Huh7 cells and transfected cells were selected. After 5 passages, stable clones were isolated to confirm plasmid entrance. Luciferase activity of the Huh7-1x-ARE-luc cell line was performed with 0-80μM lead concentration to induce oxidative stress response. Cell viability was assessed by MTT.  With Real time PCR, AREKEAP1 pathway gene expression were detected. Statistical analysis was performed by ΔCt method, using graphpad prism 6 software.
Findings: The gene expression of the reporter gene increased with increasing oxidative stress. Reducing the expression of the reporter gene was observed after 30 μM. 35 μM lead inhibited 50% cell metabolism. Expression of antioxidant pathway genes was significantly increased in 30 μM leaded cells compared to control gene.
Conclusion: The biosensor prepared from Huh7-1x-ARE-luc cell line of the reporter gene can be a convenient and efficient means for measuring oxidative compounds such as heavy metals such as lead.

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Objective: Molecular epidemiology of Visceral Leishmaniasis (VL) is currently used widely for different objectives such as vector incrimination studies. Materials and Methods: In this study three different loci including kinetoplast DNA (kDNA), ribosomal DNA (rDNA), and Cystein protease B (CPB) of Leishmania parasite genome were used for detection and identification of natural infection of sand flies of Germi district of Ardebil province, the most important VL or Kala-azar foci in Iran. Results: The results showed that the three loci of kDNA, rDNA and CPBs are respectively more appropriate for leptomonad infection/initial screening, identification of the L.donovani complex, and discrimination of the species complex. It was also verified that both members of the complex, L. donovani and L. infantum, are present in the study area and are transmitted to the hosts by

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Objective: To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties. Materials and Methods: Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining. Results: Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones. Conclusion: We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans.

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NIR Laser application in bacteria is often focused on mortality and antibiotic efficacy. The literature records on this point are absolutely diverse from mortality in different degrees to immortality and even viability enhancement. The aim of this study is to investigate 808 nm laser effects on E.coli-DH5α viability and Growth with CFU, MTT and FCM assays. To obtain the purpose, bacteria in LB media put on with 808nm laser on 100 and 200 J/cm² dosages and were investigated and compared by CFU, MTT and FCM assay. CFU assay results after 24 hours incubation were not significantly different between laser treatments and control. (P=0.06). In contrast, MTT assay results after 1 hours from laser treatment indicated significant deleterious effects in 200 J/cm² laser treatment compared with control(P=0.006). On the other hand, FCM assay results of laser treatments with using of PI and Triton X100 not only approved MTT assay results but also revealed some dose dependent changes on bacteria ranging from increase membrane permeability to lethal damages. As a conclusion of the results in these method assays, we can state that these different laser doses produce diverse effects on viability and growth in E.coli-DH5α. Consequently the laser treatments could be planned for antibiotic purposes or enhancing gene transformation process.

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Objective: The Brucella melitensis virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The product of the second gene of the virB operon, virB2, is predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in this gene, which is expected to exert polar effects on downstream genes. We researched on the evaluation of relation between virB2 mutant with immunogenicity in mouse model and intracellular replication in macrophages J774. Materials and Methods: In order to determine whether VirB2 is required for the function of the T4SS apparatus, we constructed and characterized deletion mutation of virB2 and kanamycin resistance gene replaced instead of virB2. For demonstration of intracellular replication, macrophages J774 and BALB/c mices were infected with wild type Brucella melitensis and mutated. Results: After 48 h, number of mutated Brucella severe decreased severly compaired to wild type in macrophages J774, and Brucella with virB2 deletion decreased from 1×106 CFU/spleen less than 1000 CFU/spleen during 8 weeks, also total IgG increased in both but IL-12 and IFN-γ increased only in wild type. Conclusion: VirB2 was essential for intracellular replication in mouse models and J774 macrophages. The virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus